One of the most common reasons women seek medical treatment is for vaginal discharge or other vaginal symptoms. In women who visit their physician with vaginal complaint, approximately 40% are diagnosed as having some form of vaginitis, and 90% of these cases fall into three clinical entities: bacterial vaginosis (BV), trichomoniasis, and vulvovaginal candidiasis. (See, e.g., Sobel, "Vaginal Infections in Adult Women," Medical Clinics of North America 74:1573 (1990)). The symptoms of these three distinct diseases overlap, thus creating a need for differential diagnosis before appropriate and specific medication can be prescribed. A rapid and accurate diagnosis is especially critical in pregnant women, in whom BV and trichomoniasis are associated with premature births and low birth weight babies. Moreover, BV-positive pregnant women are predisposed to chorioamnionitis, amniotic fluid infection, and puerperal infectious morbidity. BV has also been associated with pelvic inflammatory disease, postpartum endometritis, bacteremia, salpingitis, and the like.
The term "bacterial vaginosis" was coined only a few years ago, the disease being formerly known as "leukorrhea" or "non-specific" vaginitis. Until the past decade, the cause of this syndrome was presumed to be some unidentified pathogen. A study published in 1955 suggested that Gardnerella vaginalis was the causative agent of BV, but this proposition was discredited by subsequent studies revealing that G. vaginalis was present in the vaginal secretions of 10-50% of normal women, i.e., BV-negative women. Since then it has become apparent that, unlike most diseases, BV cannot be attributed to one specific etiologic agent, but instead results from a drastic alteration of the vaginal flora. The normally present Lactobacilli become greatly reduced in number, and there is a concomitant overgrowth of several anaerobic bacteria and other microorganisms, especially Gardnerella vaginalis (Gv). This alteration is accompanied by an increase in vaginal pH.
The clinical "gold standard" method of diagnosing BV involves the examination of four criteria, and does not involve microbiological culture:
1) presence of clue cells (determined microscopically); PA1 2) white or gray adherent homogeneous discharge; PA1 3) vaginal fluid pH&gt;4.5; and PA1 4) fishy amine odor when vaginal fluid is mixed with 10% potassium hydroxide (KOH). PA1 1) Obtain a vaginal swab and apply the swab to provided pH paper; PA1 2) Insert the swab into a tube containing a lysis solution comprising a low ionic strength buffer and a detergent, the lysis solution having a pH ranging from about 7.0 to about 12.0; PA1 3) Squeeze out the swab, place the tube into a well of a heating block, at heat to a temperature above about 65.degree. C. for approximately five minutes to release the nucleic acid from the microorganism in the absence of mechanical force; PA1 4) Add 5M GuSCN solution to a final concentration of 3M. PA1 5) Transfer the solution to the first sample well in an automated dipstick processor, which automatically completes the assay development in approximately 30 minutes. PA1 6) Visually determine whether a colored substance has been deposited on the bead.
To diagnose BV, some investigators require the presence of clue cells plus two of the other three indicators, while other investigators require only that any three of the four indicators be present. In practice, physicians do not typically conduct pH and amine odor tests in their offices, nor even attempt to identify clue cells. In fact, use of the gold standard test is confined primarily to clinical studies. Identification of clue cells requires special skills, since such cells are difficult to distinguish from other microscopically observable entities. Clue cells are not microorganisms, but are vaginal epithelial cells that have been shed from the vaginal wall and to which a large number of rod-shaped bacteria have adhered. The adherent cells include G. vaginalis, and other anaerobic species including, for example, Mobiluncus species.
Another consistent hallmark of BV is the elevation of vaginal pH above the normal value of 4.5. Unfortunately, this highly sensitive indicator lacks specificity, as conditions other than BV can also cause an elevated vaginal pH. For example, infection with Trichomonas vaginalis or cervicitis can cause the vaginal pH to go up. Hence, vaginal pH by itself cannot be used to diagnose BV because such a practice would result in an unacceptable incidence of false positives.
In addition to the gold standard criteria, BV is sometimes diagnosed by assessing the shift in vaginal flora by examining Gram stained vaginal smears. This method, used primarily in research protocols, is difficult to perform and requires special training, thereby rendering it unsuitable for physician's offices. Moreover, this technique is less sensitive and less specific for BV than the gold standard method. (See, e.g., Nugent, et al., "Reliability of Diagnosing Bacterial Vaginosis Is Improved By A Standardized Method of Gram Stain," J. Clin. Microbiol. 29(2):297-301 (1991).
Currently, some physicians make use of a wet mount in conjunction with office vaginal examinations. A slide prepared from the patient's vaginal fluid is visually examined by the physician. When a BV-positive patient is examined by a physician practiced in making these difficult observations, such a slide will reveal an absence of the usual levels of Lactobacilli, which are large rods, and the presence of a large number of small rod-shaped bacteria, including Gardnerella vaginalis (Gv), Prevotella, and Mobiluncus species. The former two bacteria have straight rod shapes, while the latter bacterium exhibits a curved rod shape. Some physicians believe that clue cells may be identified through wet mount analysis, but such means of identification are not generally accepted as appropriate.
When fast isolated, G. vaginalis was termed Haemophilus vaginalis. Later, G. vaginalis was reclassified as Corynebacterium vaginalis. Finally, G. vaginalis was placed into a new genus, Gardnerella, as it did not properly belong in either of the first two classifications. As such, some investigators have attempted to determine whether the amount of G. vaginalis present in a sample is indicative of BV. In doing so, they concluded that BV-positive women, on the average, have higher levels of G. vaginalis than BV-negative women. Considerable overlap was found to exist in the levels of G. vaginalis found in BV-positive and BV-negative women, however, thereby rendering the G. vaginalis cell level inconclusive evidence of the disease state. See, Amsel, et al., Am. J. Med. 74:14-22, 1983 and Eschenbach, et al., Am. J. Obstet. Gynecol. 158:819-28, 1988.
BV is one common cause of vaginal complaints. Other microorganisms commonly associated with such symptoms are Candida species and Trichomonas vaginalis. The most typical way of diagnosing candidiasis is according to symptoms, visual inspection of the vagina, and microscopic detection of the organism itself. For the wet mount, potassium hydroxide is added to dissolve epithelial cells, and the slide is examined for the presence of yeast elements, for example, pseudohyphae or budding yeast. If these measures do not yield a definitive diagnosis, the physician may order a culture. An alternative to culture method is Gram stain, which requires a trained person to analyze the results.
The classical method for the diagnosis of Trichomonas involves demonstration that the organism is present. Trichomonas is not a normal inhabitant of the vagina, and is considered a pathogen anytime it is detected. Typically, detection is done microscopically by observing protozoa with characteristic motility in vaginal secretions mixed with saline in a wet mount. Since Candida wet mounts contain potassium hydroxide, separate wet mounts must be used if one wishes to look for both of these organisms. Detection of Trichomonas depends on observation of flagellated cells of a characteristic size and shape that are in motion. Unfortunately, trichomonads quickly lose their distinctive motility upon cooling to room temperature, therefore, a microscope and trained microscopist must be available immediately after the sample is taken. Once they have lost their motility, trichomonads are practically indistinguishable from lymphocytes present on the slide. To exacerbate the challenge of microscopically detecting trichomonads is the fact that they tend to be present in low numbers.
In view of the foregoing, it is readily apparent that there are numerous disadvantages associated with the use of culture for diagnosing vaginal disorders, particularly if the woman presents with symptoms of vaginitis. The foremost disadvantage is the three to seven days required to obtain culture results. This delay can lead doctors to avoid culture altogether and, instead, to dispense medication based on a less accurate method of microscopic examination of a wet mount.
Moreover, aside from the delay in getting the results, culture can be prohibitively expensive when the syndrome can be caused by three different etiologic agents, as is the case with vaginitis. Even if a patient were willing to pay, most commercial microbiology laboratories do not offer Trichomonas vaginalis culture. Moreover, even when this culture is available, logistical problems arise from trying to culture three organisms from a single patient. If one swab is used and placed into the standard bacterial transport medium, the Trichomonas will not survive. This fastidious organism requires a specialized transport medium. Hence, at least two swabs must be taken. In fact, the microbiologist would prefer a separate swab for each organism to be cultured. Yet if three swabs are taken, it is not likely that all three will pick up identical samples, as the successive swabs are likely to deplete the vaginal fluid, and may even cause irritation.
In the case of Gardnerella vaginalis and Candida albicans, culture is of limited utility because these organisms can be present in the non-diseased vagina. In many instances, culture for these organisms would have diagnostic value if it were designed to yield quantitative data that could be used to identify clinically significant levels of these organisms, a procedure that involves plating serial dilutions of each sample. But, routine culture protocols do not involve plating serial dilutions to identify clinically significant levels and, thus, they determine only whether the organism is present. At best, the microbiology laboratory will inform the physician whether the growth was heavy or light. This limited information is not sufficient for the diagnosis of BV or candidiasis.
Even if a method were available for analyzing a single swab for the presence of multiple organisms, there are numerous drawbacks of culture and wet mount. As such, a biochemical test would be more economical than culturing for several different organisms. Moreover, if the test could be performed in less than an hour, the diagnosis could be completed before the patient left the doctor's office, thus enabling her to obtain the correct medication that same day.
One advantage of culture is that the organism is given a chance to multiply before being identified. However, since a swab can pick up only limited amounts of sample, a successful biochemical method would have to possess the capability of detecting very small numbers of organisms. As such, a biochemical method performed in the doctor's office would have to be able to yield results from the minuscule amount of sample present on one or two swabs. For tests that rely on detecting cytoplasmic components of the pathogenic organisms, the detection step must be preceded by efficient disruption of cell walls and membranes. Unfortunately, many pathogens of the vagina, e.g., Candida albicans, Gardnerella vaginalis, and Group B streptococci, are extremely difficult to lyse compared with other microorganisms. Trichomonas lyses easily, but contains potent nucleases that can easily sabotage diagnostic tests based on detection of nucleic acids.
Moreover, different methods are currently required to lyse each of these organisms. As such, the prior art has not provided a general lysis method that is effective for the simultaneous disruption and release of nucleic acids for the several pathogens of the vagina. For diagnostic tests targeted to panels rather than single microorganisms, the use of a different lysis protocol for each organism would necessitate separate swabs for each, and the separate processing would drive up the cost of the test. As a practical matter, a single lysis protocol would be far more desirable.
One potential biochemical detection method involves the use of nucleic acid hybridization. The sequence specificity embodied in nucleic acids makes it possible to differentiate virtually any two species by nucleic acid hybridization. Standard techniques for detection of specific nucleotide sequences generally employ nucleic acids that have been purified away from cellular proteins and other cellular contaminants. The most common method of purification involves lysing the cells with sodium dodecyl sulfate (SDS), digesting with proteinase K, and removing residual proteins and other molecules by extracting with organic solvents such as phenol, chloroform, and isoamylalcohol.
Endogenous nucleases released during cell solubilization can frustrate efforts to recover intact nucleic acids, particularly ribonucleic acids (RNA). While deoxyribonucleses (DNases) are easily inactivated by the addition of chelating agents to the lysis solution, ribonucleases (RNases) are far more difficult to eliminate. RNases are ubiquitous, being present even in the oil found on human hands, and they are practically indestructible. For example, the standard procedure for preparing laboratory stocks of pancreatic RNase is to boil a solution of the enzyme for 15 minutes. The purpose of this treatment is to destroy all traces of contaminating enzyme activity, since other enzymes cannot survive boiling.
Accordingly, protecting against RNase is a commonly acknowledged aspect of any standard RNA preparation technique. Sambrook, et al., which is a compendium of commonly followed laboratory practices, recommends extensive precautions to avoid RNase contamination in laboratories where RNA work is conducted. All solutions that will contact RNA are to be prepared using RNase-free glassware, autoclaved water, and chemicals reserved for work with RNA that are dispensed exclusively with baked spatulas. Besides purging laboratory reagents of RNase, RNase inhibitors are typically included in lysis solutions. These are intended to destroy endogenous RNases that generally become activated during cell lysis.
From the above descriptions, it is evident that the standard nucleic acid purification techniques are not practical for the rapid and economical detection of specific microorganisms outside of a well-equipped laboratory. Protecting against RNase is cumbersome and costly, and typical extraction procedures require the handling of caustic solvents, access to water baths, fume hoods, and centrifuges, and even the storage and disposal of hazardous wastes. The direct analysis of unfractionated solubilized microorganisms would avoid the cost and inconvenience of these purification techniques.
A minimum prerequisite for identifying microorganisms by hybridization is the release of target nucleic acids from cellular structures that otherwise would impede entry of the detection probes. Such probes consist in general of segments of nucleic acid that are complementary to sequences unique to the target organism. Once the probe has formed a hybrid with the target, the existence of that hybrid can be ascertained by activating a signal generating system that is bound to the probe.
Various impediments can block the access of hybridization probes to their target sequences, the most significant barrier being the cell wall itself. While the cell walls of many microorganisms can be effectively solubilized with guanidinium salts or with proteinase K and SDS, these methods do not effectively release readily hybridizable nucleic acids from many clinically important microorganisms, e.g., Candida albicans and Gram positive species. The Gram positive bacteria, which are known to be difficult to lyse, also do not efficiently yield hybridizable nucleic acids after treatment with guanidinium salts or proteinase K.
In some instances, unusual mounts of endogenous nucleases have aggravated the problem of recovering intact nucleic acids. For example, one of the few groups that has successfully extracted intact DNA from Trichomonas vaginalis reports that this organism is characterized by a high level of endogenous nuclease activity, and that its DNA is unusually susceptible to degradation during isolation. See, Riley, et al., J. Clin. Microbiol., 30:465-472 (1992).
Moreover, the means available for lysing recalcitrant organisms are often complex and unwieldy. For example, a common method for the mechanical lysis of yeast requires the sample to be alternately vortexed with glass beads and cooled in an ice bath. The cellular extract is recovered by centrifugation after puncturing the bottom of the tube. Similarly, a Mini-Beadbeater.TM. has been used for lysing Mycobacterium species, where cells are ruptured by vigorous shaking with phenol and zirconium beads. See, Hurley, et al., Journal of Clinical Microbiology, 25:2227-2229 (1987).
The lysis of soil bacteria presents another challenge that has required drastic measures. Successful methods for their lysis have included multiple cycles of freeze-thawing, and passage through a French press, which is a high-pressure shearing device. One recent method for lysing these bacteria calls for the successive application of sonication, microwave heating, and thermal shocks. See, Picard, et al., Applied and Environmental Microbiology, 58:2717-2722 (1992).
Another common approach for lysis of microorganisms has involved enzymes that attack the cell walls. For example, lyticase has proven effective in lysing Candida albicans, while achromopeptidase, mutanolysin, or proteinase K removes cell walls from most Gram positive microorganisms. See, e.g., Kaneko, et al., Agr. Biol. Chem., 37:2295-2302 (1973); Bollet, et al., Nucleic Acids Research, 19:1955 (1991); Siegel, et al., Infection and Immunity, 31:808-815 (1981). However, the use of enzymes in routine detection protocols is fraught with disadvantages. Chief among these is cost, but calibration of stock solutions, lengthy incubation times, the need for low temperature storage, and limited shelf life also make the use of enzymes less than desirable for protocols involving rapid detection of microorganisms.
When the microorganisms to be detected are located in human clinical samples, additional concerns must be accommodated. For one, the presence of mucous can cause clinical samples from some sources to be viscous and unmanageable. A successful lysis procedure must disperse mucous and any other substances that may accompany the sample. Furthermore, the method of lysis must be compatible with conventional sampling techniques if they are to be widely accepted by the medical community. For example, samples from the vagina are customarily taken with a single cotton or dacron swab. Therefore, samples available for detection of vaginal pathogens normally will be limited to whatever material that can be eluted from such a swab.
In view of the foregoing, there exists a need for a simple and rapid method for releasing intact nucleic acid from both prokaryotic and eukaryotic microorganisms present in a single, biological sample. Moreover, there exists a need for a simple, fast and effective biochemical method which selectively detects the microorganisms associated with vaginitis, i.e., Gardnerella vaginalis, Trichomonas vaginalis and Candida albicans. The present invention remedies these needs by providing such methods.